Intracellular thiol-disulfide balance is critical for the activity of proteins with functionally important cysteine residues. The tripeptide glutathione (GSH) and oxidoreductases like glutaredoxins (GRXs) and thioredoxins (TRX) help maintain thiol-disulfide balance by catalyzing disulfide exchange reactions and protecting cysteines residues from oxidation by reactive oxygen species (ROS). Mitochondrial GSH metabolism is a key component of cellular thiol redox homeostasis since this organelle is the main source and target of ROS produced from aerobic metabolism. Essential mitochondrial functions such as oxidative phosphorylation, protein import, and Fe-S cluster biogenesis are directly dependent on thiol-disulfide balance. Consequently, disruption of mitochondrial thiol-disulfide balance has been linked to cancer, neurodegenerative diseases, and aging. The long-term objective of this research program is to characterize mitochondrial thiol redox homeostasis using the yeast Saccharomyces cerevisiae as a model system. The mitochondrion is divided by a double membrane into two distinct compartments: the matrix and the intermembrane space (IMS). Redox regulatory systems that govern thiol-disulfide balance in the matrix are well-characterized. However, the mechanisms for thiol-disulfide redox control in the IMS and the role of GSH metabolism in IMS thiol redox pathways represent key gaps in our knowledge of mitochondrial redox systems. The goal of this proposal is to determine the mechanisms for maintaining thiol-disulfide balance in the IMS. GFP-based redox sensors that are targeted to the IMS and matrix have been developed to allow localized thiol redox monitoring via in vivo fluorescence measurements. These sensors equilibrate with the local reduced/oxidized glutathione (GSH:GSSG) pool and register thiol redox changes via disulfide bond formation. To determine how GSH metabolism influences the IMS redox environment, GSH:GSSG exchange between the IMS, matrix, and cytosol under redox stress will be characterized using these sensors and mitochondrial GSH transporters will be identified (Aim 1). Furthermore, the connection between GSH metabolism and thiol- dependent protein import into the IMS will be deciphered by determining how alterations in GSH:GSSG or the IMS protein import machinery influence each other (Aim 2). Finally, additional thiol redox systems/pathways that help maintain IMS thiol redox balance will be identified (Aim 3). By using a molecular genetics approach in a highly tractable organism, these studies will shed new light on thiol redox control pathways in the IMS, with strong implications for disorders involving mitochondrial dysfunction.